The goal of this project is to understand how mRNAs, proteins, and subcellular organelles are distributed and organized in muscle and nerve cells. In multinucleated muscle cells, the nonuniform distribution of specific mRNAs and proteins contributes to the formation of the neuromuscular junction. In neurons, differential distribution of specific mRNAs may play a role into the establishment and maintenance of axonal-dendritic polarity. We are trying to understand how mRNA stability and mRNA translation influence mRNA localization. To that effect, we are localizing, by in situ hybridization, both endogenous mRNAs and foreign mRNAs from transfected genes, in the mouse muscle cell line C2, and in primary cultures of rat hippocampal neurons. In the past year we have started to consider the role of translation in mRNA localization in neurons, taking as model the mRNA for ferritin, whose translation is regulated by the level of iron in the milieu. After specific inhibition of ferritin, mRNA translation is by a decrease in free iron, ferritin mRNA is found in both cell body. These results suggest that translation of ferritin mRNA is involved in its perinuclear localization. In the future we will continue to investigate the role of this parameter. We are continuing to investigate the mechanism of vesicle and protein traffic in muscle by studying the localization of the glucose transporter GLUT4 and its translocation to the plasma membrane following stimulation by insulin or exercise. A detailed study of GLUT4 localization in C2 myotubes has been carried out. GLUT4 and several markers of subcellular organelles were localized by single and double immunofluorescence in control myotubes and in myotubes treated with the fungal metabolite Brefeldin A. The results suggest that GLUT4 is stored in vesicles that are specific and distinct from those that carry other markers of the endosomal-lysosomal pathway. These results are significant because specificity of the GLUT4 storage compartment has long been a subject of debate.